Wednesday, August 11, 2021 5:11:00 AM
1). “a linker connecting said first and second nucleic acid sequences in a manner that allows both the said first nucleic acid sequence to hybridize to the target and extend on the 3' end and the second nucleic acid sequences to hybridize to the target at the same time;”
2). “The present invention also relates to cooperatively linked nucleic acids that also comprise a probe. This modified primer/probe is similar to the cooperative nucleic acid, but with the addition of one or more detectable labels to either the capture sequence or the primer, turning it into a probe. Because extension of the cooperative primer/probe is detectable, it can be useful in a variety of applications including multiplexing applications that require differentiation of SNP's using an ARMS based approach. In some embodiments, both the primer and the probe are designed with Tm's below the melting temperature which is used in the amplification reaction, so that the primer will not amplify without the probe binding and the probe will not have a signal without the primer binding. This creates two points of specificity in the same primer/probe combination.
The cooperative nucleic acids, such as primers and probes, of this invention are useful in a variety of primer extension/amplification reactions known to those skilled in the art, including, but not limited to the polymerase chain reaction, rolling circle amplification, nucleic acid sequencing and others. The cooperative primers and probes of this invention can also be used in applications that have post extension/amplification steps, such as hybridization to an array. Because the cooperative primers/probes in this invention substantially reduce primer-dimers, they are of particular use in multiplexed and highly multiplexed reactions.
The use of a cooperative nucleic acid can decrease the amount of primer-dimer present by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent compared to the amount of primer-dimer present when a normal primer (a non-cooperative nucleic acid) is used.
Therefore, disclosed herein is a cooperative nucleic acid molecule comprising: a) a first nucleic acid sequence, wherein the first nucleic acid sequence is substantially complementary to a first region of a target nucleic acid, and wherein the first nucleic acid sequence is extendable on the 3' end; b) a second nucleic acid sequence, wherein the second nucleic acid sequence is substantially complementary to a second region of the target nucleic acid; wherein the first and second nucleic acid sequences are attached to each other; and wherein the second nucleic acid sequence hybridizes to the target nucleic acid sequence downstream from the 3' end of the first nucleic acid sequence; and wherein the effective melting temperature (Tm) of the first nucleic acid molecule is increased by at least 1° C. as compared to the isolated Tm of the first nucleic acid sequence without the second nucleic acid sequence attached to it.
The cooperative nucleic acid may be linear or circularized.
By “extendable on the 3' end” is meant that the first nucleic acid is free on this end to be amplified, or extended. This is meant to include heat activatable primers such as those described by Lebedev et al, among other technologies.
The first nucleic acid sequence is a primer, and the second nucleic acid sequence is alternatively referred to as a “capture nucleic acid sequence.” Either the first or the second sequence may have a detectable label, or a third sequence may have a detectable label. The first and second nucleic acid sequences can be attached via a linker, which can be a non-nucleic acid sequence. In one example, the linker can attach the 5' end of the first nucleic acid sequence to the 3' end of the second nucleic acid sequence. This can be seen, for instance, in FIG. 2. Alternatively, the the first nucleic acid sequence is inverted such that the 5' end of the first nucleic acid sequence is attached to the 5' end of the second nucleic acid sequence. This can be seen, for instance, in FIG. 3. In yet another example, the 5' end of the second nucleic acid sequence can be linked to the first nucleic acid sequence in the middle of the sequence, as seen in FIG. 1. It is noted that by “middle of the sequence” is meant that the linker is not joined to the first nucleic acid sequence at either the 5' end or the 3' end of the nucleic acid, but rather is attached to a nucleotide internal to the nucleotides on the 5' and 3' ends.
In one example, the cooperative nucleic acid comprises 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or less continuous nucleotides in the same orientation. In other words, this is the number of nucleotides that are part of a single, unbroken nucleic acid sequence and oriented in the same 5' to 3' direction, or the 3' to 5' direction. By way of example, if the linker is a nucleic acid sequence, it can include the linker, if the nucleotides in the linker are in the same orientation as either the first or second nucleic acid sequence to which it is directly connected.
The linker can be made of nucleic acids, non-nucleic acids, or some combination of both. If the linker is made of nucleic acids, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, or 100 or more nucleotides in length, or any number in between. Types of linkers are discussed elsewhere herein. The linker can be any length, and can be longer or shorter than the combined length of the first and second nucleic acid sequences, longer or shorter than just the first nucleic acid sequence, or longer or shorter than the second nucleic acid sequence.
Furthermore, there can be a space on the target nucleic acid where the first nucleic acid sequence and the second nucleic acid sequence hybridize. In other words, there are two distinct regions on the target nucleic acid, one which hybridizes with the first nucleic acid sequence, and the other which hybridizes to the second nucleic acid sequence. The distance between the first and second regions on the target can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, or 100 or more nucleotides in length.”
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